Protein phosphorylation is now well recognized as an important mechanism utilized by cells to transduce and regulate signals during different stages of cellular function (Hunter, Phil. Trans. R. Soc. Lond. B 353: 583–605 (1998); Chan et al., Annu. Rev. Immunol. 12: 555–592 (1994); Zhang, Curr. Top. Cell. Reg. 35: 21–68 (1997); Matozaki and Kasuga, Cell. Signal. 8: 113–19 (1996); Fischer et al, Science 253:401–6 (1991); Flint et al., EMBO J. 12:1937–46 (1993)). There are at least two major classes of phosphatases: (1) those that dephosphorylate proteins (or peptides) that contain a phosphate group(s) on a serine or threonine moiety (termed Ser/Thr phosphatases) and (2) those that remove a phosphate group(s) from the amino acid tyrosine (termed protein tyrosine phosphatases or PTPases or PTPs).
The PTPases are a family of enzymes that can be classified into two groups: a) intracellular or nontransmembrane PTPases and b) receptor-type or transmembrane PTPases.
Intracellular PTPases: Most known intracellular type PTPases contain a single conserved catalytic phosphatase domain consisting of 220–240 amino acid residues. The regions outside the PTPase domains are believed to play important roles in localizing the intracellular PTPases subcellularly (Mauro, L. J. and Dixon, J. E. TIBS 19: 151–155 (1994)). The first intracellular PTPase to be purified and characterized was PTP-1B, which was isolated from human placenta (Tonks et al., J. Biol. Chem. 263: 6722–6730 (1988)). Shortly after, PTP1B was cloned (Charbonneau et al, Proc. Natl. Acad. Sci. USA 86: 5252–5256 (1989); Chernoff et al., Proc. Natl. Acad. Sci. USA 87: 2735–2789 (1989)). Other examples of intracellular PTPases include (1) T-cell PTPase/TC-PTP (Cool et al. Proc. Natl. Acad. Sci. USA 86: 5257–5261 (1989)), (2) rat brain PTPase (Guan et al., Proc. Natl. Acad. Sci. USA 87: 1501–1502 (1990)), (3) neuronal phosphatase STEP (Lombroso et al., Proc. Natl Sci. USA 88: 7242–7246 (1991)), (4) ezrin-domain containing PTPases: PTPMEG1 (Guet al., Proc. Natl. Acad. Sci. USA 88: 5867–57871 (1991)), PTPH1 (Yang and Tonks, Proc. Natl. Acad. Sci. USA 88: 5949–5953 (1991)), PTPD1 and PTPD2 (Møller et al, Proc. Natl. Acad. Sci. USA 91: 7477–7481 (1994)), FAP-1/BAS (Sato et al., Science 268: 411–415 (1995); Banville et al., J. Biol. Chem. 269: 22320–22327 (1994); Maekawa et al., FEBS Letters 337: 200–206 (1994)), and SH2 domain containing PTPases: PTP1C/SH-PTP1/SHP-1 (Plutzky et al., Proc. Natl. Acad. Sci. USA 89:1123–1127 (1992); Shen et al., Nature Lond. 352: 736–739 (1991)) and PTP1D/Syp/SH-PTP2/SHP-2 (Vogel et al., Science 259: 1611–1614 (1993); Feng et al., Science 259: 1607–1611 (1993); Bastein et al., Biochem. Biophys. Res. Comm. 196:124–133 (1993)).
Receptor-type PTPases consist of a) a putative ligand-binding extracellular domain, b) a transmembrane segment, and c) an intracellular catalytic region. The structures and sizes of the putative ligand-binding extracellular domains of receptor-type PTPases are quite divergent. In contrast, the intracellular catalytic regions of receptor-type PTPases are very homologous to each other and to the intracellular PTPases. Most receptor-type PTPases have two tandemly duplicated catalytic PTPase domains.
The first receptor-type PTPases to be identified were (1) CD45/LCA (Ralph, S. J., EMBO J. 6: 1251–1257 (1987)) and (2) LAR (Streuli et al., J. Exp. Med. 168: 1523–1530 (1988)) that were recognized to belong to this class of enzymes based on homology to PTP1 B (Charbonneau et al., Proc. Natl. Acad. Sci. USA 86: 5252–5256 (1989)). CD45 is a family of high molecular weight glycoproteins and is one of the most abundant leukocyte cell surface glycoproteins and appears to be exclusively expressed upon cells of the hematopoietic system (Trowbridge and Thomas, Ann. Rev. Immunol. 12: 85–116 (1994)).
The identification of CD45 and LAR as members of the PTPase family was quickly followed by identification and cloning of several different members of the receptor-type PTPase group. Thus, 5 different PTPases, (3) PTPα, (4) PTPβ, (5) PTPδ, (6) PTPε, and (7) PTPζ, were identified in one early study (Krueger et al., EMBO J. 9: 3241–3252 (1990)). Other examples of receptor-type PTPases include (8) PTPγ (Barnea et al., Mol. Cell. Biol. 13: 1497–1506 (1995)) which, like PTPζ(Krueger and Saito, Proc. Natl. Acad. Sci. USA 89: 7417–7421 (1992)) contains a carbonic anhydrase-like domain in the extracellular region, (9) PTPμ (Gebbink et al., FEBS Letters 290: 123–130 (1991)), (10) PTPκ (Jiang et al., Mol. Cell. Biol. 13: 2942–2951 (1993)). Based on structural differences the receptor-type PTPases may be classified into subtypes (Fischer et al., Science 253: 401–406 (1991)): (I) CD45; (II) LAR, PTPδ, (11) PTPσ; (III) PTP□, (12) SAP-1 (Matozaki et al., J. Biol. Chem. 269: 2075–2081 (1994)), (13) PTP-U2/GLEPP1 (Seimiya et al., Oncogene 10: 1731–1738 (1995); Thomas et al., J. Biol. Chem. 269: 19953–19962 (1994)), and (14) DEP-1; (IV) PTPα, PTPε. All receptor-type PTPases except Type III contain two PTPase domains. Novel PTPases are continuously identified. In the early days of PTPase research, it was believed that the number of PTPs would match that of protein tyrosine kinases (PTKs) (Hanks and Hunter, FASEB J. 9: 576–596 (1995)). However, although about 90 open reading frames in C. elegans contain the hallmark motif of PTPs, it now seems that the estimate of ‘classical’ PTPases must be downsized, perhaps to between 100 and 200 in humans.
PTPases are the biological counterparts to protein tyrosine kinases Therefore, one important function of PTPases is to control, down-regulate, the activity of PTKs. However, a more complex picture of the function of PTPases has emerged. Thus, several studies have shown that some PTPases may actually act as positive mediators of cellular signaling. As an example, the SH2 domain-containing SHP-2 seems to act as a positive mediator in insulin-stimulated Ras activation (Noguchi et al., Mol. Cell. Biol. 14: 6674–6682 (1994)) and of growth factor-induced mitogenic signal transduction (Xiao et al., J. Biol. Chem. 269: 21244–21248 (1994)), whereas the homologous SHP-1 seems to act as a negative regulator of growth factor-stimulated proliferation (Bignon and Siminovitch, Clin. Immunol. Immunopathol. 73: 168–179 (1994)). Another example of PTPases as positive regulators has been provided by studies designed to define the activation of the Src-family of tyrosine kinases. In particular, several lines of evidence indicate that CD45 is positively regulating the activation of hematopoietic cells, possibly through dephosphorylation of the C-terminal tyrosine of Fyn and Lck (Chan et al., Annu. Rev. Immunol. 12: 555–592 (1994)).
PTPases were originally identified and purified from cell and tissue lysates using a variety of artificial substrates and, therefore, their natural function of dephosphorylation was not well known. Since tyrosine phosphorylation by tyrosine kinases is usually associated with cell proliferation, cell transformation and cell differentiation, it was assumed that PTPases were also associated with these events. This association has now been proven to be the case with many PTPases. PTP1 B, a phosphatase whose structure was the first PTPase to be elucidated (Barford et al., Science 263:1397–1404 (1994)) has been shown to be involved in insulin-induced oocyte maturation (Flint et al., The EMBO J. 12:1937–46 (1993)) and it has been suggested that the overexpression of this enzyme may be involved in p185c-erb B2 associated breast and ovarian cancers (Wiener, et al., J. Natl. cancer Inst. 86:372–8 (1994); Weiner et al, Am. J. Obstet. Gynecol. 170:1177–883 (1994)). The association with cancer is recent evidence which suggests that overexpression of PTP1 B is statistically correlated with increased levels of p185c-erb B2 in ovarian and breast cancer. The role of PTP1 B in the etiology and progression of the disease has not yet been elucidated. Inhibitors of PTP1 B may therefore help clarify the role of PTP1 B in cancer and in some cases provide therapeutic treatment for certain forms of cancer.
PTPases: The Insulin Receptor Signaling Pathway/Diabetes
Insulin is an important regulator of different metabolic processes and plays a key role in the control of blood glucose. Defects related to its synthesis or signaling lead to diabetes mellitus. Binding of insulin to the insulin receptor (IR) causes rapid (auto)phosphorylation of several tyrosine residues in the intracellular part of the β-subunit. Three closely positioned tyrosine residues (the tyrosine-1150 domain) must all be phosphorylated to obtain full activity of the insulin receptor tyrosine kinase (IRTK) which transmits the signal further downstream by tyrosine phosphorylation of other cellular substrates, including insulin receptor substrate-1 (IRS-1) (Wilden et al., J. Biol. Chem. 267:16660–16668 (1992); Myers and White, Diabetes 42: 643–650 (1993); Lee and Pilch, Am. J. Physiol. 266: C319–C334 (1994); White et al., J. Biol. Chem. 263: 2969–2980 (1988)). The structural basis for the function of the tyrosine-triplet has been provided by X-ray crystallographic studies of IRTK that showed tyrosine-1150 to be autoinhibitory in its unphosphorylated state (Hubbard et al., Nature 372: 746–754 (1994)) and of the activated IRTK (Hubbard, EMBO J. 16:5572–5581 (1997)).
Several studies clearly indicate that the activity of the auto-phosphorylated IRTK can be reversed by dephosphorylation in vitro (reviewed in Goldstein, Receptor 3: 1–15 (1993); Mooney and Anderson, J. Biol. Chem. 264: 6850–6857 (1989)), with the tri-phosphorylated tyrosine-1150 domain being the most sensitive target for protein-tyrosine phosphatases (PTPases) as compared to the di- and mono-phosphorylated forms (King et al., Biochem. J. 275: 413418 (1991)). This tyrosine-triplet functions as a control switch of IRTK activity and the IRTK appears to be tightly regulated by PTP-mediated dephosphorylation in vivo (Khan et al., J. Biol. Chem. 264: 12931–12940 (1989); Faure et al., J. Biol. Chem. 267: 11215–11221 (1992); Rothenberg et al., J. Biol. Chem. 266: 8302–8311 (1991)). The intimate coupling of PTPases to the insulin signaling pathway is further evidenced by the finding that insulin differentially regulates PTPase activity in rat hepatoma cells (Meyerovitch et al., Biochemistry 31: 10338–10344 (1992)) and in livers from alloxan diabetic rats (Boylan et al., J. Clin. Invest. 90: 174–179 (1992)).
Until recently, relatively little was known about the identity of the PTPases involved in IRTK regulation. However, the existence of PTPases with activity towards the insulin receptor can be demonstrated as indicated above. Further, when the strong PTPase-inhibitor pervanadate is added to whole cells an almost full insulin response can be obtained in adipocytes (Fantus et al., Biochemistry 28: 8864–8871 (1989); Eriksson et al., Diabetologia 39: 235–242 (1995)) and skeletal muscle (Leighton et al., Biochem. J. 276: 289–292 (1991)). In addition, other studies show that a new class of peroxovanadium compounds act as potent hypoglycemic compounds in vivo (Posner et al.,supra). Two of these compounds were demonstrated to be more potent inhibitors of dephosphorylation of the insulin receptor than of the EGF-receptor, thus indicating that even such relatively unselective inhibitors may convey some specificity in regulating different signal transduction pathways.
It was recently found by Montreal-based research groups that mice lacking the protein tyrosine phosphatase-1B gene (PTP1B) (Elchebly et al., Science 283: 1544–1548 (1999)) yielded healthy mice that showed increased insulin sensitivity and resistance to diet-induced obesity. Importantly, these results have been confirmed and extended independently by another research team from Boston (Klaman et al., Mol. Cell. Biol. 20: 5479–5489 (2000)). The enhanced insulin sensitivity of the PTP−/+ mice was also evident in glucose and insulin tolerance tests. The PTP-1B knock-out mouse showed many characteristics which would be highly desirable to have for an anti-diabetes treatment. Most importantly, the knock-out mice grew normally and were fertile and have exhibited no increased incidence of cancer, as obviously there could have been concerns when one considers the mitogenic properties of insulin. From the diabetes perspective, the first notable features of the knock-out animals were that blood glucose and insulin levels were lowered, and the consequent marked increase in insulin sensitivity in the knock-out animals. Moreover, the insulin-stimulated tyrosine phosphorylation levels of IR and IRS-1 were found to be increased/prolonged in muscle and liver—but not in fat tissue. Thus, the main target tissues for this type of approach would appear to be insulin action in liver and muscle. This is in contrast to the main target tissue for the PPARγ agonist class of insulin sensitizers (the “-diones”), which is adipose tissue (Murphy & Nolan, Exp. Opin. Invest. Drugs 9: 1347–1361 (2000)). Several other “diabetic” parameters were also improved, such as plasma triglycerides being decreased in the knockout mice. However, perhaps even more remarkably and unexpectedly, the knock-out animals also exhibited a resistance to weight gain when placed on a high-fat diet. This is again in contrast to the action of the PPARγ agonist class of insulin sensitizers, which rather induce weight gain (Murphy & Nolan, supra), and would suggest that inhibition of PTP-1 B could be a particularly attractive option for treatment of obese Type II diabetics. This is also supported by the fact that the heterozygous mice from this study showed many of these desirable features. In the Montreal study, there appeared to be no gender differences, whereas in the Boston study in general the male animals had larger responses to PTP-1 B being knocked out. In both studies, the reduction in weight gain of the knock-out animals on the high fat diet was found to be due to a decreased fat cell mass, although differences were observed with respect to fat cell number. Leptin levels were also lower in the knock-out mice, presumably as a reflection of the decreased fat mass. Significantly, the Boston group also found that the knock-out animals had an increased energy expenditure of around 20% and an increased respiratory quotient compared to the wild-type; again, heterozygote animals displayed an intermediate level of energy expenditure. Whether this increase in metabolic rate is a reflection of the effects of PTP-1 B on insulin-signaling or on other cellular components remains to be established, but the bottom-line message that inhibition of this enzyme may be an effective anti-diabetic and perhaps also anti-obesity therapy is clear.
It should also be noted that in the PTP-1 B knock-out mice the basal tyrosine phosphorylation level of the insulin receptor tyrosine kinase does not appear to be increased, which is in contrast to the situation after insulin treatment where there is an increased or prolonged phosphorylation. This might indicate that other PTPs are controlling the basic phosphorylation state of the insulin receptor in the knock-out mice—and perhaps in man.
Previous findings are in accordance with the results reported by Elchebly et al. (supra) (recently reviewed in Kennedy, Biomed. Pharmacother. 53: 466–470 (1999)). Thus, it has been found that high glucose concentration induce insulin resistance and increase the expression of PTP1B in rat (fibroblasts expressing the human insulin receptor (Maegawa et al., J. Biol. Chem. 270: 7724–7730 (1995)). In rat L6 cells, insulin and insulin-like growth factor I (IGF-1) were found to induce increased PTPase activity, including increased PTP1 B expression (Kenner et al., J. Biol. Chem. 266: 25455–25462 (1993)). In addition, the same group has shown that PTP1 B may interact directly with the activated IR (Seely et al. Diabetes 45:1379–1385 (1996)) and act directly as a negative regulator of insulin and IGF-1-stimulated signaling (Kenner et a. J. Biol. Chem. 271: 19810–19816 (1996)). Osmotic loading of rat KRC-7 hepatoma cells with neutralizing anti-PTP1 B antibodies also indicated a role for PTP1 B in negative regulating of the insulin signaling pathway (Akmad et al. J. Biol. Chem. 270: 20503–20508 (1995)).
Also other PTPases have been implicated as regulators of the insulin signaling pathway. Thus, it was found that the ubiquitously expressed SH2 domain containing PTPase, PTP1 D/SHP-2 (Vogel et al., 1993, supra), associates with and dephosphorylates IRS-1, but apparently not the IR itself (Kuhné et al., J. Biol. Chem. 268: 11479–11481 (1993); (Kuhné et al., J. Biol. Chem. 269: 15833–15837 (1994)).
Other studies suggest that receptor-type or membrane-associated PTPases are involved in IRTK regulation (Faure et al., J. Biol. Chem. 267: 11215–11221 (1992), (Haring et al., Biochemistry 23: 3298–3306 (1984); Sale, Adv. Prot. Phosphatases 6: 159–186 (1991)). Hashimoto et al. have proposed that LAR might play a role in the physiological regulation of insulin receptors in intact cells (Hashimoto et al., J. Biol. Chem. 267: 13811 –13814 (1992)). Their conclusion was reached by comparing the rate of dephosphorylation/Inactivation of purified IR using recombinant PTP1 B as well as the cytoplasmic domains of LAR and PTPα. Antisense inhibition was used to study the effect of LAR on insulin signaling in a rat hepatoma cell line (Kulas et al., J. Biol. Chem. 270: 2435–2438 (1995)). A suppression of LAR protein levels by about 60 percent was paralleled by an approximately 150 percent increase in insulin-induced auto-phosphorylation. However, only a modest 35 percent increase in IRTK activity was observed, whereas the insulin-dependent phosphatidylinositol 3-kinase (PI 3-kinase) activity was significantly increased by 350 percent. Reduced LAR levels did not alter the basal level of IRTK tyrosine phosphorylation or activity. The authors speculate that LAR could specifically dephosphorylate tyrosine residues that are critical for PI 3-kinase activation either on the insulin receptor itself or on a downstream substrate. Conflicting results have been reported for PTP-LAR knock-out mice. Thus, Goldstein and coworkers reported that transgenic mice deficient in PTP-LAR exhibit profound defects in glucose-homeostasis (Ren et al., Diabetes 47: 493–497 (1998)). However, it is difficult to fully assess the contribution of LAR deficiency to the glucose homeostasis in these mice due to the fact that the control mice were of a different genetic background than the knock-out mice. Moreover, normal glucose homeostasis was reported in a different strain of PTP-LAR knock-out mice (Sorensen et al., Diabetologia 40: A143 (1997)).
While previous reports indicate a role of PTPα in signal transduction through src activation (Zheng et al., Nature 359: 336–339 (1992); den Hertog et al., EMBO J. 12: 37893798 (1993)) and interaction with GRB-2 (den Hertog et al., EMBO J. 13: 3020–3032 (1994); Su et al., J. Biol. Chem. 269: 18731–18734 (1994)), Møller, Lammers and coworkers provided results that suggest a function for this phosphatase and its close relative PTP□ as negative regulators of the insulin receptor signal (Møller et al., 1995 supra; Lammers, et al., FEBS Lett. 404:37–40 (1997). These studies also indicated that receptor-like PTPases might play a significant role in regulating the IRTK.
Other studies have shown that PTP1 B and TC-PTP are likely to be involved in the regulation of several other cellular processes in addition to the described regulatory roles in insulin signaling. Therefore, PTP1B and/or TC-PTP as well as other PTPases showing key structural features with PTP1 B and TC-PTP are likely to be important therapeutic targets in a variety of human and animal diseases. The compounds of the present invention are useful for modulating or inhibiting PTP1 B and/or TC-PTP and/or other PTPases showing key structural features with said PTPases and for treating diseases in which said modulation or inhibition is indicated. A few examples that are not intended in any way to limit the scope of the invention of substrates that may be regulated by PTP1 B will be given below.
Tonks and coworkers have developed an elegant ‘substrate trapping’ technique that has allowed identification of the epidermal growth factor receptor (EGF-R) as a major substrate of PTP1B in COS cells (Flint et al. Proc. Natl. Acad. Sci. USA 94: 1680–1685 (1997)). In addition, three other as yet unidentified substrates of PTP1 B were isolated. As an example of these studies, it has been found—using the above substrate-trapping technique—that PTP1 B in addition to the EGF-R associates with activated platelet-derived growth factor receptor (PDGF-R), but not with colony-stimulating factor 1 receptor (CSF-1R) (Liu & Chernoff, Biochem. J. 327: 139–145 (1997)).
Early studies have shown that the subcellular localization as well as the enzyme activity of PTP1 B may be regulated by agonist-induced calpain-catalyzed cleavage in human platelets (Frangioni et al. EMBO J. 12: 4843–4856 (1993)). Moreover, PTP1 B cleavage correlated with the transaction from reversible to irreversible platelet aggregation. Thus, as a non-limiting example compounds of the present invention might be used to prevent or induce irreversible platelet aggregation in individuals in need thereof. It was proposed that the cleavage-induced change in the subcellular localization of PTP1 B (from membrane to cytosol) results in different substrate specificity not only in platelet but also in other cell types (Frangioni et al., supra).
The above substrate trapping method has further been used to identify the protein tyrosine kinase p210bcr-abl as a substrate for PTP1 B (LaMontagne, Jr. et al. Mol. Cell. Biol. 18: 2965–2975 (1998)). These studies suggest that PTP1 B might function as a negative regulator of p210bcr-abl signaling in vivo. In addition, PTP1B was recently found to bind to and dephosphorylate the docking protein p130 Cas in rat fibroblasts and hereby suppress transformation by v-crk, v-src, and v-ras, but not by v-raf (Liu et al. Mol. Cell. Biol. 18: 250–259 (1998)).
The transmembrane PTPase CD45, which is believed to be hematopoietic cell-specific, was found to negatively regulate the insulin receptor tyrosine kinase in the human multiple myeloma cell line U266 (Kulas et al., J. Biol. Chem. 271: 755–760 (1996)).
Further, PTPases influences the following hormones or diseases or disease states: somatostatin, the immune system/autoimmunity, cell—cell interactions/cancer, platelet aggregation, osteoporosis, and microorganisms, as disclosed in PCT Publication WO 99/15529.
Somatostatin inhibits several biological functions including cellular proliferation (Lamberts et al., Molec. Endocrinol. 8: 1289–1297 (1994)). While part of the antiproliferative activities of somatostatin are secondary to its inhibition of hormone and growth factor secretion (e.g. growth hormone and epidermal growth factor), other antiproliferative effects of somatostatin are due to a direct effect on the target cells. As an example, somatostatin analogs inhibit the growth of pancreatic cancer presumably via stimulation of a single PTPase, or a subset of PTPases, rather than a general activation of PTPase levels in the cells (Liebow et al., Proc. Natl. Acad. Sci. USA 86: 2003–2007 (1989); Colas et al., Eur. J. Biochem. 207:1017–1024 (1992)).
PTPases: the Immune System/Autoimmunity
Several studies suggest that the receptor-type PTPase CD45 plays a critical role not only for initiation of T cell activation, but also for maintaining the T cell receptor-mediated signaling cascade. These studies are reviewed in: (Weiss A., Ann. Rev. Genet. 25: 487510 (1991); Chan et al., Annu. Rev. Immunol. 12: 555–592 (1994); Trowbridge and Thomas, Annu. Rev. Immunol. 12: 85–116 (1994)).
CD45 is one of the most abundant of the cell surface glycoproteins and is expressed exclusively on hemopoetic cells. In T cells, it has been shown that CD45 is one of the critical components of the signal transduction machinery of lymphocytes. In particular, evidence has suggested that CD45 phosphatase plays a pivotal role in antigen-stimulated proliferation of T lymphocytes after an antigen has bound to the T cell receptor (Trowbridge, Ann. Rev. Immunol, 12: 85–116 (1994)). Several studies suggest that the PTPase activity of CD45 plays a role in the activation of Lck, a lymphocyte-specific member of the Src family protein-tyrosine kinase (Mustelin et al., Proc. Natl. Acad. Sci. USA 86: 6302–6306 (1989); Ostergaard et al., Proc. Natl. Acad. Sci. USA 86: 8959–8963 (1989)). These authors hypothesized that the phosphatase activity of CD45 activates Lck by dephosphorylation of a C-terminal tyrosine residue, which may, in turn, be related to T-cell activation. Thus, it was found that recombinant p56lck specifically associates with recombinant CD45 cytoplasmic domain protein, but not to the cytoplasmic domain of the related PTPα (Ng et al., J. Biol. Chem. 271: 1295–1300 (1996)). The p56lck-CD45 interaction seems to be mediated via a nonconventional SH2 domain interaction not requiring phosphotyrosine. In immature B cells, another member of the Src family protein-tyrosine kinases, Fyn, seems to be a selective substrate for CD45 compared to Lck and Syk (Katagiri et al., J. Biol. Chem. 270: 27987–27990 (1995)).
Studies using transgenic mice with a mutation for the CD45-exon6 exhibited lacked mature T cells. These mice did not respond to an antigenic challenge with the typical T cell mediated response (Kishihara et al., Cell 74:143–56 (1993)). Inhibitors of CD45 phosphatase would therefore be very effective therapeutic agents in conditions that are associated with autoimmune diseases with rheumatoid arthritis, systemic lupus erythematosus, type I diabetes, and inflammatory bowel disease as non-limiting examples. Another important use of CD45 inhibitors is for immunosuppression in connection with tissue or cell transplantation and other condtions with need for immunosuppressive treatment.
CD45 has also been shown to be essential for the antibody mediated degranulation of mast cells (Berger et al., J. Exp. Med. 180:471–6 (1994)). These studies were also done with mice that were CD45-deficient. In this case, an igE-mediated degranulation was demonstrated in wild type but not CD45-deficient T cells from mice. These data suggest that CD45 inhibitors could also play a role in the symptomatic or therapeutic treatment of allergic disorders with asthma, allergic rhinitis, food allergy, eczema, urticaria and anaphylaxis as nonlimiting examples.
Another PTPase, an inducible lymphoid-specific protein tyrosine phosphatase (HePTP) has also been implicated in the immune response. This phosphatase is expressed in both resting T and B lymphocytes, but not non-hemopoetic cells. Upon stimulation of these cells, mRNA levels from the HePTP gene increase 10–15 fold (Zanke et al., Eur. J. Immunol. 22: 235–239 (1992)). In both T and B cells HePTP may function during sustained stimulation to modulate the immune response through dephosphorylation of specific residues. Its exact role, however remains to be defined.
Likewise, the hematopoietic cell specific SHP-1 seems to act as a negative regulator and play an essential role in immune cell development. In accordance with the above-mentioned important function of CD45, HePTP and SHP-1, selective PTPase inhibitors may be attractive drug candidates both as immunosuppressors and as immunostimulants. Recent studies illustrate the potential of PTPase inhibitors as immunomodulators by demonstrating the capacity of the non-selective vanadium-based PTPase inhibitor, BMLOV, to induce apparent B cell selective apoptosis compared to T cells (Dawson et al., FEBS Lett. 478: 233 –236; Schieven et al., J. Biol. Chem. 270: 20824–20831 (1995)).
PTPases: Cell—Cell Interactions/Cancer
Focal adhesion plaques, an in vitro phenomenon in which specific contact points are formed when fibroblasts grow on appropriate substrates, seem to mimic, at least in part, cells and their natural surroundings. Several focal adhesion proteins are phosphorylated on tyrosine residues when fibroblasts adhere to and spread on extracellular matrix (Gumbiner, Neuron 11: 551–564 (1993)). However, aberrant tyrosine phosphorylation of these proteins can lead to cellular transformation. The intimate association between PTPases and focal adhesions is supported by the finding of several intracellular PTPases with ezrin-like N-terminal domains, e.g. PTPMEG1 (Gu et al, Proc. Natl. Acad. Sci. USA 88: 5867–5871 (1991), PTPH1 (Yang and Tonks, Proc. Natl. Acad. Sci. USA 88: 5949–5953 (1991)) and PTPD1 (Møller et al., Proc. Natl. Acad. Sci. USA 91: 7477–7481 (1994)). The ezrin-like domain shows similarity to several proteins that are believed to act as links between the cell membrane and the cytoskeleton. PTPD1 was found to be phosphorylated by and associated with c-src in vitro and is hypothesized to be involved in the regulation of phosphorylation of focal adhesions (Møller et al., supra).
PTPases may oppose the action of tyrosine kinases, including those responsible for phosphorylation of focal adhesion proteins, and may therefore function as natural inhibitors of transformation. TC-PTP, and especially the truncated form of this enzyme (Cool et al., Proc. Natl. Acad. Sci. USA 87: 7280–7284 (1990)), can inhibit the transforming activity of v-erb and v-fms (Lammers et al., J. Biol. Chem. 268: 22456–22462 (1993), Zander et al., Oncogene 8: 1175–1182 (1993)). Moreover, it was found that transformation by the oncogenic form of the HER2/neu gene was suppressed in NIH 3T3 fribroblasts overexpressing PTP1 B (Brown-Shimer et al., Cancer Res. 52: 478482 (1992)).
The expression level of PTP1 B was found to be increased in a mammary cell line transformed with neu (Zhay et al., Cancer Res. 53: 2272–2278 (1993)). The intimate relationship between tyrosine kinases and PTPases in the development of cancer is further evidenced by the finding that PTPε is highly expressed in murine mammary tumors in transgenic mice over-expressing c-neu and v-Ha-ras, but not c-myc or int-2 (Elson and Leder, J. Biol. Chem. 270: 26116–26122 (1995)). Further, the human gene encoding PTPγ was mapped to 3p21, a chromosomal region, which is frequently deleted in renal and lung carcinomas (LaForgia et al., Proc. Natl. Acad. Sci. USA 88: 5036–5040 (1991)).
In this context, it seems significant that PTPases appear to be involved in controlling the growth of fibroblasts. Thus, it was found that Swiss 3T3 cells harvested at high density contain a membrane-associated PTPase whose activity on an average is 8-fold higher than that of cells harvested at low or medium density (Pallen and Tong, Proc. Natl. Acad. Sci. USA 88: 6996–7000 (1991)). It was hypothesized by the authors that density-dependent inhibition of cell growth involves the regulated elevation of the activity of the PTPase(s) in question. In accordance with this view, a membrane-bound, receptor-type PTPase, DEP-1, showed enhanced (>=10-fold) expression levels with increasing cell density of WI-38 human embryonic lung fibroblasts and in the AG1518 fibroblast cell line (Ostman et al., Proc. Natl. Acad. Sci. USA 91: 9680–9684 (1994)).
Two closely related receptor-type PTPases, PTPκ and PTPμ, can mediate homophilic cell—cell interaction when expressed in non-adherent insect cells, suggesting that these PTPases might have a normal physiological function in cell-to-cell signalling (Gebbink et al., J. Biol. Chem. 268: 16101–16104 (1993), Brady-Kalnay et al., J. Cell Biol. 122: 961–972 (1993); Sap et al., Mol. Cell. Biol. 14: 1–9 (1994)). Interestingly, PTPκ and PTPμ do not interact with each other, despite their structural similarity (Zondag et al., J. Biol. Chem. 270: 14247–14250 (1995)). From the studies described above it is apparent that PTPases may play an important role in regulating normal cell growth. However, as pointed out above, other studies indicate that PTPases may also function as positive mediators of intracellular signaling and thereby induce or enhance mitogenic responses. Increased activity of certain PTPases might therefore result in cellular transformation and tumor formation. Indeed, in one study over-expression of PTPμ was found to lead to transformation of rat embryo fibroblasts (Zheng, supra). In addition, SAP-1 was found to be highly expressed in pancreatic and colorectal cancer cells. SAP-1 is mapped to chromosome 19 region q13.4 and might be related to carcinoembryonic antigen mapped to 19q13.2 (Uchida et al., J. Biol. Chem. 269: 12220–12228 (1994)). Further, the dsPTPase, cdc25, dephosphorylates cdc2 at Thr14/Tyr-15 and thereby functions as positive regulator of mitosis (reviewed by Hunter, Cell 80: 225–236 (1995)). Inhibitors of specific PTPases are therefore likely to be of significant therapeutic value in the treatment of certain forms of cancer.
PTPases: Platelet Aggregation
PTPases seem to be centrally involved in platelet aggregation. Thus, agonist-induced platelet activation results in calpain catalyzed cleavage of PTP1B with a concomitant 2-fold stimulation of PTPase activity (Frangioni et al., EMBO J. 12: 4843–4856 (1993)). The cleavage of PTP1 B leads to subcellular relocation of the enzyme and correlates with the transition from reversible to irreversible platelet aggregation in platelet-rich plasma. In addition, the SH2 domain containing PTPase, SHP-1, was found to translocate to the cytoskeleton in platelets after thrombin stimulation in an aggregation-dependent manner (Li et al., FEBS Lett 343: 89–93 (1994)).
Although some details in the above two studies have been questioned, there is over-all agreement that PTP1 B and SHP-1 play significant functional roles in platelet aggregation (Ezumi et al., J. Biol. Chem. 270: 11927–11934 (1995)). In accordance with these observations, treatment of platelets with the PTPase inhibitor pervanadate leads to significant increase in tyrosine phosphorylation, secretion and aggregation (Pumiglia et al., Biochem. J. 286: 441449 (1992)).
PTPases: Osteoporosis
The rate of bone formation is determined by the number and the activity of osteoblasts, which in term are determined by the rate of proliferation and differentiation of osteoblast progenitor cells, respectively. Histomorphometric studies indicate that the osteoblast number is the primary determinant of the rate of bone formation in humans (Gruber et al., Mineral Electrolyte Metab. 12: 246–254 (1987), reviewed in Lau et al., Biochem. J. 257: 2336 (1989)). Acid phosphatases/PTPases may be involved in negative regulation of osteoblast proliferation. Thus, fluoride, which has phosphatase inhibitory activity, has been found to increase spinal bone density in osteoporotics by increasing osteoblast proliferation (Lau et al., supra). Consistent with this observation, an osteoblastic acid phosphatase with PTPase activity was found to be highly sensitive to mitogenic concentrations of fluoride (Lau et al., J. Biol. Chem. 260: 4653–4660 (1985), Lau et al., J. Biol. Chem. 262: 1389–1397 (1987), Lau et al., Adv. Protein Phosphatases 4: 165–198 (1987)). Interestingly, the level of membrane-bound PTPase activity was increased dramatically when the osteoblast-like cell line UMR 106.06 was grown on collagen type-I matrix compared to uncoated tissue culture plates. Since a significant increase in PTPase activity was observed in density-dependent growth arrested fibroblasts (Pallen and Tong, Proc. Natl. Acad. Sci. 88: 6996–7000 (1991)), it might be speculated that the increased PTPase activity directly inhibits cell growth The mitogenic action of fluoride and other phosphatase inhibitors (molybdate and vanadate) may thus be explained by their inhibition of acid phosphatases/PTPases that negatively regulate the cell proliferation of osteoblasts. The complex nature of the involvement of PTPases in bone formation is further suggested by the identification of a novel parathyroid regulated, receptor-like PTPase, OST-PTP, expressed in bone and testis (Mauro et al., J. Biol. Chem. 269: 30659–30667 (1994)). OST-PTP is up-regulated following differentiation and matrix formation of primary osteoblasts and subsequently down-regulated in the osteoblasts which are actively mineralizing bone in culture. It may be hypothesized that PTPase inhibitors may prevent differentiation via inhibition of OST-PTP or other PTPases thereby leading to continued proliferation. This would be in agreement with the above-mentioned effects of fluoride and the observation that the tyrosine phosphatase inhibitor orthovanadate appears to enhance osteoblast proliferation and matrix formation (Lau et al., Endocrinology 116: 2463–2468 (1988)). In addition, it was observed that vanadate, vanadyl and pervanadate all increased the growth of the osteoblast-like cell line UMR106. Vanadyl and pervanadate were stronger stimulators of cell growth than vanadate. Only vanadate was able to regulate the cell differentiation as measured by cell alkaline phosphatase activity (Cortizo et al., Mol. Cell. Biochem. 145: 97–102 (1995)). It is of particular interest to the current invention that several studies have shown that bisphosphonates, such as alendronate and tiludronate, inhibit the PTPase activity in osteoclasts, and that the inhibition of PTPase activity correlated with the inhibition of in vitro osteoclast formation and boneresorption (Schmidt et al., Proc. Natl. Acad. Sci. U.S.A. 93: 3068–3073 (1996); Murakami et al., Bone 20: 399404 (1997); Opas et al., Biochem. Pharmacol. 54: 721–727 (1997); Skorey et al., J. Biol. Chem. 272: 22472–22480 (1997)). Thus, PTPase inhibitors other than bisphophonates—can potentially be effective for prevention and/or treatment of osteoporosis.
PTPases: Microorganisms
Dixon and coworkers have called attention to the fact that PTPases may be a key element in the pathogenic properties of Yersinia (reviewed in Clemens et al. Molecular Microbiology 5: 2617–2620 (1991)). This finding was rather surprising since tyrosine phosphate is thought to be absent in bacteria. The genus Yersinia comprises 3 species: Y. pestis (responsible for the bubonic plague), Y. pseudoturberculosis and Y. enterocolitica (causing enteritis and mesenteric lymphadenitis). Interestingly, a dual-specificity phosphatase, VH1, has been identified in Vaccinia virus (Guan et al., Nature 350: 359–253 (1991)). These observations indicate that PTPases may play critical roles in microbial and parasitic infections, and they further point to PTPase inhibitors as a novel, putative treatment principle of infectious diseases.
WO 99/46267 discloses compounds, which are pharmacologically useful inhibitors of PTPases. However, the present invention, which represents a novel selection under WO 99/46267, discloses a class of compounds which surprisingly are more potent against protein tyrosine phosphatases (e.g. PTP1 B) than those disclosed in WO 99/46267.